Ionomycin Research Articles

Palmitoleic Acid Extracted from Fish Oil Inhibit aGVHD By Immune Regulation

Objective: This study aims to explore the immunosuppressive effect of palmitoleic acid (POA), derived from fish oil extract, on lymphocytes in vitro and its potential impact on the clinical outcome and survival prognosis of rodents with acute graft-versus-host disease (aGVHD). Methods: In vitro experiments involved using CCK8 to determine the influence of various concentrations of POA on the growth and proliferation of lymphocytes. Flow cytometry was employed to assess POA's inhibitory effect on T lymphocyte activation triggered by fluorobol ester (PMA) and ionomycin (IM). For evaluating the therapeutic effect of POA on aGVHD, C57bl/c mice were used as donors and BALB/C mice as recipients. The aGVHD mouse model was established to observe the impact of POA on aGVHD treatment, survival, and prognosis. Results: In vitro experiments demonstrated that POA concentrations ranging from 250uM to 500uM inhibited the proliferation of PMA and IM-activated T lymphocytes and reduced the proportion of CD4+CD25+ cells without causing significant cytotoxicity. The aGVHD mouse model exhibited clinical symptoms like bristling, depilation, anal redness and swelling, and loose stools, along with characteristic pathological changes in the liver and small intestine. Treatment with POA significantly extended the survival period and reduced the clinical score of aGVHD in the model mice. Conclusion: Palmitoleic acid derived from fish oil shows promise as a potential treatment for acute graft-versus-host disease following allogeneic hematopoietic stem cell transplantation, warranting further research on its immunosuppressive properties. Keywords: Fish oil extract, palmitoleic acid, acute graft-versus-host disease, allogeneic hematopoietic stem cell transplantation, immunosuppression.

Generation of reporter mice for detecting the transcriptional activity of nuclear factor of activated T cells.

Nuclear factor of activated T cells (NFAT) is a transcription factor essential for immunological and other biological responses. To develop analyzing system for NFAT activity in vitro and in vivo, we generated reporter mouse lines introduced with NFAT-driven enhanced green fluorescent protein (EGFP) expressing gene construct. Six tandem repeats of -286 to -265 of the human IL2 gene to which NFAT binds in association with its co-transcription factor, activator protein (AP)-1, was conjunct with thymidine kinase minimum promoter and following EGFP coding sequence. Upon introduction of the resulting reporter cassette into C57BL/6 fertilized eggs, the transgenic mice were obtained. Among 7 transgene-positive mice in 110 mice bone, 2 mice showed the designated reporter mouse character. Thus, the EGFP fluorescence of CD4+ and CD8+ T cells in these mice was enhanced by stimulation through CD3 and CD28. Each of phorbol 12-myristate 13-acetate (PMA) and ionomycin (IOM) stimulation weakly but their combined stimulation strongly enhanced EGFP expression. The stimulation-induced EGFP upregulation was also observed following T cell subset differentiation in a different manner. The EGFP induction by PMA + IOM stimulation was more potent than that by CD3/CD28 stimulation in helper T (Th)1, Th2, Th9, and regulatory T cells, while both stimulation conditions displayed the equivalent EGFP induction in Th17 cells. Our NFAT reporter mouse lines are useful for analyzing stimulation-induced transcriptional activation mediated by NFAT in cooperation with AP-1 in T cells.

Hochu-ekki-to enhanced airway ciliary beating by an [Ca2+]i increase via TRPV4 in mice

BackgroundHochu-ekki-to (TJ-41, a traditional herbal medicine) is used for preventing airway infections caused by viruss or bacterias in patients with chronic obstructive pulmonary disease (COPD). TJ-41 is known to inhibit viral adhesion and to activate mucosal immune system. However, the effects of TJ-41 on the beating cilia, which sweep away inhaled viruses and bacteria from the airways (mucociliary clearance, a host defense mechanism of lungs), still remain uncertain. Hypothesis/purposeTJ-41 may activate the airway ciliary beating, activation of which increases the rate of mucociliary clearance leading to inhibit airway infections. MethodsTJ-41 was orally administered to mice for 4–8 weeks (1.7 - 1.8 g/kg/day) and airway ciliary cells were isolated from the lungs. The frequency (CBF) and angle (CBA, an index of amplitude) of ciliary beating were measured using the high-speed video microscopy (HSVM, 500 fps), in relation to intracellular Ca2+ concentration ([Ca2+]i). Differences were considered significant p<0.05. ResultsThe TJ-41 treatment enhanced the basal CBF and CBA of airway cilia by 10% mediated via an increase in [Ca2+]i. Moreover, ionomycin (IM) at 1 nM increased CBF and CBA by elevating [Ca2+]i in airway ciliary cells from TJ-41 treated mice, but never in those from non-TJ-41 treated mice. These increases in [Ca2+]i were caused by the enhancement of temperature-dependent Ca2+ entry pathways, including transient receptor potential vanilloid 4 (TRPV4). ConclusionTJ-41 enhanced the basal CBF and CBA of airway cilia via a temperature-dependent [Ca2+]i increase and it also enhanced increases in CBF, CBA and [Ca2+]i stimulated by 1 nM IM, which never increased CBF and CBA in cells from non-treated mice. Thus, TJ-41 enhanced the temperature-dependent Ca2+ entry pathways including TRPV4. The [Ca2+]i increase, a novel action of TJ-41, may induce various pharmacological activities already reported including activation of the airway ciliary beating. These results suggest the potential of therapeutic use for patients suffering from chronic disease to improve various activities in addition to prevention of airway infections.

Neutrophil Extracellular Traps Are Increased in Chronic Myeloid Leukemia and Are Differentially Affected by Tyrosine Kinase Inhibitors.

Simple SummaryNeutrophil extracellular traps (NETs) are a recently described form of neutrophil cellular death that has been associated with a thrombotic tendency in many diseases. We studied NET formation in neutrophils derived from patients with chronic myeloid leukemia (CML) and in CML neutrophil cell lines and demonstrated that NETs are increased in CML and that certain drugs used to treat CML (tyrosine kinase inhibitors—TKIs) increase NET formation. These findings may shed light on a novel mechanism linking CML, TKIs and vascular toxicity.Cardiovascular complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML). We studied neutrophil extracellular trap (NET) formation in CML and evaluated the effect of TKIs on NET formation. Neutrophils isolated from treatment-naïve patients with CML showed a significant increase in NET formation compared to matched controls at baseline and after stimulation with ionomycin (IO) and phorbol 12-myristate 13-acetate (PMA). Expression of citrullinated histone H3 (H3cit), peptidyl arginine deiminase 4 (PAD4) and reactive oxygen species (ROS) was significantly higher in CML samples compared to controls. Pre-treatment of neutrophils with TKIs was associated with a differential effect on NET formation, and ponatinib significantly augmented NET-associated elastase and ROS levels as compared to controls and other TKIs. BCR-ABL1 retroviral transduced HoxB8-immortalized mouse hematopoietic progenitors, which differentiate into neutrophils in-vitro, demonstrated increased H3cit & myeloperoxidase (MPO) expression consistent with excess NET formation. This was inhibited by Cl-amidine, a PAD4 inhibitor, but not by the NADPH inhibitor diphenyleneiodonium (DPI). Ponatinib pre-exposure significantly increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with IO. In summary, CML is associated with increased NET formation, which is augmented by ponatinib, suggesting a possible role for NETs in promoting vascular toxicity in CML.

Small Molecule Inhibitors Targeting Nuclear Factor κB Activation Markedly Reduce Expression of Interleukin-2, but Not Interferon-γ, Induced by Phorbol Esters and Calcium Ionophores.

The T-box transcription factor Eomesodermin (Eomes) promotes the expression of interferon-γ (IFN-γ). We recently reported that the small molecule inhibitors, TPCA-1 and IKK-16, which target nuclear factor κB (NF-κB) activation, moderately reduced Eomes-dependent IFN-γ expression in mouse lymphoma BW5147 cells stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In the present study, we investigated the direct effects of NF-κB on IFN-γ expression in mouse lymphoma EL4 cells and primary effector T cells. Eomes strongly promoted IFN-γ expression and the binding of RelA and NFATc2 to the IFN-γ promoter when EL4 cells were stimulated with PMA and IM. Neither TPCA-1 nor IKK-16 reduced IFN-γ expression; however, they markedly decreased interleukin (IL)-2 expression in Eomes-transfected EL4 cells. Moreover, TPCA-1 markedly inhibited the binding of RelA, but not that of Eomes or NFATc2 to the IFN-γ promoter. In effector CD4+ and CD8+ T cells activated with anti-CD3 and anti-CD28 antibodies, IFN-γ expression induced by PMA and A23187 was not markedly decreased by TPCA-1 or IKK-16 under conditions where IL-2 expression was markedly reduced. Therefore, the present results revealed that NF-κB is dispensable for IFN-γ expression induced by PMA and calcium ionophores in EL4 cells expressing Eomes and primary effector T cells.

CML-271: Neutrophil Extracellular Traps Are Increased in Chronic Myeloid Leukemia and Are Differentially Affected by Tyrosine Kinase Inhibitors

Context: Cardiovascular (CVS) complications are increasingly reported with the use of certain tyrosine kinase inhibitors (TKIs) to treat chronic myeloid leukemia (CML). Neutrophil extracellular traps (NETs) have previously been implicated in the pathogenesis of cancer-associated thrombosis. Objective: To assess NET formation in neutrophils from patients with CML, to characterize the role of reactive oxygen species (ROS) and peptidyl arginine deiminase 4 (PAD4) in NET formation in CML, and to assess the effects of TKIs on NET formation in CML. Design: Blood samples were obtained from patients with newly diagnosed CML and from age/gender-matched controls. NETs were assessed after stimulation with phorbol 12-myristate 13-acetate (PMA) or ionomycin (IO), with or without pre-treatment with imatinib, ponatinib, nilotinib, and dasatinib at clinically relevant concentrations. NET formation was assessed by morphology, immunofluorescence (IF), and the expression of proteins known to be associated with NET formation (NET-associated elastase, citrullinated histone H3 [H3cit], etc.). ER-HoxB8 conditionally immortalized murine myeloid progenitor cell line was differentiated into functional neutrophils and transduced with a BCR-ABL1 oncogene (or empty vector control). Results: Neutrophils isolated from treatment-naive patients with CML showed a significant increase in NET formation compared to matched controls at baseline and after stimulation with IO as assessed by IF and after stimulation with PMA as assessed by NET-associated elastase expression. Expression of H3cit, PAD4, and ROS was significantly higher in CML samples compared to controls. Pre-treatment of neutrophils with TKIs was associated with a differential effect on NET formation. Exposure to ponatinib significantly augmented NET-associated elastase and ROS levels as compared to control and other TKIs in neutrophils derived from patients with CML. BCR-ABL1 retrovirally transduced HoxB8-neutrophils demonstrated increased H3cit and myeloperoxidase expression, consistent with excess NET formation. This was inhibited by Cl-amidine, a PAD4 inhibitor, but not by the NADPH inhibitor diphenyleneiodonium (DPI). Ponatinib and nilotinib pre-exposure significantly increased H3cit expression in HoxB8-BCR-ABL1 cells after stimulation with IO. Conclusions: CML is associated with increased NET formation, which is further augmented by ponatinib and nilotinib, suggesting a possible role for NETs in promoting vascular toxicity in CML. In the ER-HoxB8-BCR-ABL1 model, NET formation is PAD4- but not ROS-dependent.

Superoxide Dismutase 3 Controls the Activation and Differentiation of CD4+T Cells.

Superoxide dismutase 3 (SOD3), a well-known antioxidant has been shown to possess immunomodulatory properties through inhibition of T cell differentiation. However, the underlying inhibitory mechanism of SOD3 on T cell differentiation is not well understood. In this study, we investigated the effect of SOD3 on anti-CD3/CD28- or phorbol myristate acetate (PMA) and ionomycin (ION)-mediated activation of mouse naive CD4+ T cells. Our data showed that SOD3 suppressed the expression of activation-induced surface receptor proteins such as CD25, and CD69, and cytokines production. Similarly, SOD3 was found to reduce CD4+T cells proliferation and suppress the activation of downstream pathways such as ERK, p38, and NF-κB. Moreover, naïve CD4+T cells isolated from global SOD3 knock-out mice showed higher expression of CD25, CD69, and CD71, IL-2 production, proliferation, and downstream signals compared to wild-type CD4+T cells. Whereas, the use of DETCA, a known inhibitor of SOD3 activity, found to nullify the inhibitory effect of SOD3 on CD4+T cell activation of both SOD3 KO and wild-type mice. Furthermore, the expression of surface receptor proteins, IL-2 production, and downstream signals were also reduced in Th2 and Th17 differentiated cells upon SOD3 treatment. Overall, our data showed that SOD3 can attenuate CD4+T cell activation through modulation of the downstream signalings and restrict CD4+T cell differentiation. Therefore, SOD3 can be a promising therapeutic for T cell-mediated disorders.

Eomesodermin promotes interaction of RelA and NFATc2 with the Ifng promoter and multiple conserved noncoding sequences across the Ifng locus in mouse lymphoma BW5147 cells

The T-box transcription factor Eomesodermin (Eomes) regulates the lineage-dependent expression of interferon γ (IFN-γ). We previously showed that Eomes promotes IFN-γ production and interacts with multiple conserved noncoding sequences (CNS) across the Ifng locus in mouse lymphoma BW5147 cells. In the present study, we investigated the transcriptional regulation of IFN-γ by the nuclear factor κB (NF-κB) subunit RelA and nuclear factor of activated T cells c2 (NFATc2, also known as NFAT1) in Eomes-transfected BW5147 cells. Eomes promoted the interaction of RelA and NFATc2 with the Ifng promoter and five CNS, including CNS-22 and CNS+30 upon stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). The dual NF-κB and STAT3 inhibitor TPCA-1 moderately reduced the PMA- and IM-induced IFN-γ transcription in Eomes-transfected BW5147 cells. TPCA-1 interfered with RelA binding to the Ifng promoter, CNS-22 and CNS+30. Moreover, TPCA-1 reduced the interaction of Eomes or NFATc2 with the Ifng promoter and CNS+30. The present results indicate that Eomes promotes the interaction of RelA and NFATc2 with the Ifng promoter and multiple CNS across the Ifng locus in BW5147 cells.

Structure-Effect Relationships of Novel Semi-Synthetic Cannabinoid Derivatives.

Background: As a library of cannabinoid (CB) derivatives with (-)-trans-cannabidiol (CBD) or (-)-trans-cannabidivarin (CBDV) scaffold, we synthesized nine novel cannabinoids: 2-hydroxyethyl cannabidiolate (2-HEC), 2-hydroxypentyl cannabidiolate (2-HPC), 2,3-dihydroxypropyl cannabidiolate (GCBD), cyclohexyl cannabidiolate (CHC), n-hexyl-cannabidiolate (HC), 2-(methylsulfonamido)ethyl cannabidiolate (NMSC), 2-hydroxyethyl cannabidivarinolate (2-HECBDV), cyclohexyl cannabidivarinolate (CHCBDV), and n-hexyl cannabidivarinolate (HCBDV). Their binding and intrinsic effects at the CB1- and CB2-receptors and the effects on inflammatory signaling cascades were investigated in in vitro and ex vivo cell models. Materials and Methods: Binding affinity was studied in membranes isolated from CB-receptor-transfected HEK293EBNA cells, intrinsic functional activity in Chinese hamster ovary (CHO) cells, and activation of nuclear factor κB (NF-κB) and nuclear factor of activated T-cells (NFAT) in phorbol 12-myristate 13-acetate (PMA)/ionomycin (IO)-treated Jurkat T-cells. Inhibition of interleukin (IL)-17-induced pro-inflammatory cytokines and chemokines [IL-6, IL-1β, CC-chemokine ligand 2 (CCL2), and tumor necrosis factor (TNF)-α] was studied in RAW264.7 macrophages at the RNA level. Pro-inflammatory cytokine (IL-1β, IL-6, IL-8, and TNF-α) expression and prostaglandin E2 (PGE2) expression were investigated at the protein level in lipopolysaccharide (LPS)-treated primary human monocytes. Results: Derivatives with long aliphatic side chains at the ester position at R1 [HC (5)] as well as the ones with polar side chains [2-HECBDV (7), NMSC (6), and 2-HEC (1)] can be selective for CB2-receptors. The CBDV-derivatives HCBDV and CHCBDV demonstrated specific binding at CB1- and CB2-receptors at nanomolar concentrations. 2-HEC, 2-HPC, GCBD, and NMSC were agonists at CB2-receptor and antagonists at CB1-receptor. CHC bound both receptors at submicromolar ranges and was an agonist for these receptors. 2-HECBDV was an agonist at CB2-receptor and an antagonist at the CB1-receptor despite its modest affinity at this receptor (micromolar range). NMSC inhibited NF-κB and NFAT activity, and 2-HEC, 2-HPC, and GCBD dose-dependently inhibited PMA/IO-stimulated NFAT activation. CHC and HC dose-dependently reduced IL-1β and CCL2 messenger RNA (mRNA) expression. NMSC inhibited IL-1β, CCL2, and TNF-α at lower doses. At higher doses, it induced a pronounced increase in IL-6 mRNA. 2-HEC, 2-HPC, and GCBD dose-dependently inhibited LPS-induced IL-1β, TNF-α, and IL-6 synthesis. NMSC further increased LPS-stimulated IL-1β release but inhibited IL-8, TNF-α, and PGE2. Conclusion: The CBD- and CBDV-derivatives studied are suitable for targeting CB-receptors. Some may be used as selective CB2 agonists. The length of the aliphatic rest at R2 of CBD (pentyl) and CBDV (propyl) did not correlate with the binding affinity. Higher polarity at R1 appeared to favor the agonistic activity at CB2-receptors.

Functional Change of Effector Tumor-Infiltrating CCR5+CD38+HLA-DR+CD8+ T Cells in Glioma Microenvironment.

Human glioma facilitates an impaired anti-tumor immunity response, including defects in circulation of T lymphocytes. The level of CD8+ T-cell activation acts as an immune regulator associated with disease progression. However, little is known about the characteristics of peripheral and tumor-infiltrating CD8+ T cells in patients with glioma. In this study, we examined the level of CD8+ T-cell activation in a group of 143 patients with glioma and determined that peripheral CD3+ T cells decreased in accordance with disease severity. The patients' peripheral CD8+ T-cell populations were similar to that of healthy donors, and a small amount of CD8+ tumor-infiltrating lymphocytes was identified in glioma tissues. An increase in activated CD8+ T cells, characterized as CD38+HLA-DR+, and their association with disease progression were identified in the patients' peripheral blood and glioma, and shown to display enriched CCR5+ and TNFR2+ expression levels. Ex vivo examination of CD38+HLA-DR+CD8+ T cells indicated that this subset of cells displayed stronger secretion of IFN-γ and IL-2 before and after a 6-h stimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin (ION) relative to healthy CD38+HLA-DR+CD8+ T cells, indicating the functional feasibility of CD38+HLA-DR+CD8+ T cells. Higher CCL5 protein and mRNA levels were identified in glioma tissues, which was consistent with the immunohistochemistry results revealing both CCL5 and CD38+HLA-DR+CD8+ T cell expression. Patients' CCR5+CD38+HLA-DR+CD8+ T cells were further validated and shown to display increases in CD45RA+CCR7− and T-bet+ accompanied by substantial CD107-a, IFN-γ, and Granzyme B levels in response to glioma cells.

Therapeutic effects of baicalin on lupus nephritis in a lupus-prone mouse model and its regulatory role in follicular helper T cell differentiation

Objective To evaluate the therapeutic effect of baicalin on lupus nephritis in a lupus-prone mouse model, and to investigate its regulatory role in the differentiation of follicular helper T (Tfh) cells. Methods Eight 12-week-old female MRL/lpr lupus-prone mice were randomly and equally divided into two groups by a random number table i.e., baicalin group and control group intraperitoneally injected with 200 mg/kg baicalin sodium and chloride physiological solution, respectively, once every day for 4 weeks. After the end of treatment, Coomassie brilliant blue staining was performed to detect the level of 24-hour urine protein. Then, the mice were sacrificed, and the spleens were resected and weighed. Mononuclear cells were isolated from these spleens, and flow cytometry was conducted to determine the proportion of Tfh cells. Additionally, the kidneys were resected and subjected to hematoxylin and eosin (HE) staining for the evaluation of kidney impairment. Moreover, some other mononuclear cells were isolated from the spleens of the lupus-prone mice in the control group, and magnetic activated cell sorting (MACS) was performed to isolate naive CD4+ T cells, which were divided into 3 groups: blank control group receiving no treatment, induction group treated with 10 μg/L anti-interleukin (IL) -21 and anti-IL-6 antibodies and 3 μg/L anti-CD3 and anti-CD28 antibodies for 5 days, and intervention group additionally treated with 40 μmol/L baicalin for 5 days besides the above treatment. Then, 50 μg/L phorbol ester, 750 μg/L ionomycin and 20 mg/L brefeldin A were used to stimulate some cultured naive CD4+ T cells in the above groups. Flow cytometry was conducted to determine the proportion of CD4+CXCR5+PD-1+ cells and CD4+IL-21+ cells. Statistical analysis was carried out with SPSS20.0 software by using one-way analysis of variance (ANOVA) and student t test for the comparison of quantitative data between groups. Results The baicalin treatment could effectively improve the kidney impairment in the lupus-prone mice. Compared with the control group, the baicalin group showed significantly decreased 24-hour urine protein level ([1 416 ± 171] vs. [2 623 ± 278] μg/24 h, P = 0.022) , and significantly decreased proportion of Tfh cells in the spleen (12.6% ± 2.3% vs. 40.2% ± 1.1%, P = 0.005) . In vitro baicalin could further inhibit the differentiation of Tfh cells. Compared with the induction group, the intervention group showed significantly decreased proportion of CD4+CXCR5+PD-1+ Tfh cells (13.3% ± 0.8% vs. 17.6% ± 0.9%, P = 0.04) and CD4+IL-21+ cells (1.0% ± 0.4% vs. 2.7% ± 0.2%, P < 0.01) . Conclusion Baicalin can effectively ameliorate lupus nephritis, which may be associated with the inhibition of Tfh cell differentiation. Key words: Lupus erythematosus, systemic; Lupus nephritis; Baicalin; Disease models, animal; Follicular helper T cells; Interleukin-21

Optimization of parthenogenetic activation of rabbit oocytes and development of rabbit embryo by somatic cell nuclear transfer

The present study explored a suitable parthenogenetic activation (PA) procedure for rabbit oocytes and investigated the developmental potential of somatic cell nuclear transfer (SCNT) embryos using rabbit foetal fibroblasts (RFFs). The electrical activation had the optimal rate of blastocyst (14.06%) when oocytes were activated by three direct current (DC) pulses (40V/mm, 20μs each) followed by 6-dimethylaminopurine (6-DMAP) and cycloheximide (CHX) treatment; the blastocyst rate of ionomycin (ION)+6-DMAP+CHX (12.07%) activation was higher than that of ION+6-DMAP (8.6%) activation or ION+CHX (1.24%) activation; there was no significant difference in blastocyst rate between ION+6-DMAP+CHX and DC+6-DMAP+CHX groups. The blastocyst rate of ION+6-DMAP+CHX-activated oocytes in the basic rabbit culture medium (M-199)+10% foetal bovine serum (FBS; 14.28%) was higher than that in buffalo conditioned medium (5.75%) or G1/G2 medium (0), and the blastocyst rate was increased when M-199+10% FBS was supplemented with amino acids. Refreshing culture medium every day or every other day significantly increased the blastocyst rate. Treatment of donor cells with 0.5% FBS for 3-5days increased blastocyst rate of SCNT embryos (33.33%) than no serum starvation (22.47%) or 0.5% FBS treatment for 6-9days (23.61%); the blastocyst rate of SCNT embryos derived from nontransgenic RFFs was higher than that derived from transgenic RFFs by electroporation. The blastocyst development ability of SCNT embryos derived from RFFs by electroporation (32.22%) was higher than that of liposome (19.11%) or calcium phosphate (20.00%) transfection, and only the embryos from electroporation group have the EGFP expression (24.44%). In conclusion, this study for the first time systematically optimized the conditions for yield of rabbit embryo by SCNT.

Effects of different activation protocols on cleavage rate and blastocyst production of caprine oocytes.

The present study was undertaken to assess the effect of different chemical activators along with 6-DMAP on in vitro matured caprine oocytes. From 4332 ovaries, 14235 cumulus oocyte complexes (COCs) were collected which were matured in TCM-199 medium containing follicle stimulating hormone (FSH) (5 µg/ml), Leutinizing hormone (LH) (10 µg/ml), oestradiol-17β (1 µg/ml) supplemented with 10% fetal bovine serum, 10% follicular fluid and 3 mg/ml bovine serum albumin (BSA) at 38.5°C and 5% CO2 in an incubator under humidified air for 27 h. In group 1 (control), 3117 in vitro matured oocytes were co incubated with sperms for 18 h in ferttalp medium. In group 2, 3563 in vitro matured oocytes were activated with 7% ethanol for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. In group 3, 3109 in vitro matured oocytes were activated with 5 μM ionomycin for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. In group 4, 3455 in vitro matured oocytes were activated with 5 μM calcium ionophore for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h in mCR2aa medium. Oocytes were cultured in 50 µL drops of research vitro cleave (RVCL) medium for embryo development. The cleavage rate, morula and blastocyst production in group 1, 2, 3 and 4 were 26.07 ± 2.37%, 14.91 ± 2.91 & 1.45 ± 0.71%, 49.57 ± 3.79%, 20.07 ± 2.38% & 5.29 ± 1.42%, 50.18 ± 3.59%, 15.26 ± 2.87% & 1.85 ± 0.72% and 80.26 ± 2.30%, 35.33 ± 2.67 & 7.10 ± 0.89%, respectively. These results indicated that the activation of in vitro matured oocytes by 5 μM calcium ionophore for 5-7 min followed by treatment with 2.0 mM DMAP for 4 h is most favorable for parthenogenetic caprine embryos production.

Nuclear translocation and function of Calcyclin (S100A6)-binding-protein/Siah-1 interacting protein in response to intracellular calcium concentration by ionomycin in SW480 cells

Objective To exploring the nuclear translocation of calcyclin(S100A6)-binding-protein (CacyBP)/SIP in response to elevated intracellular calcium concentration ([Ca2+ ]i) by ionomycin in SW480 cells. And the effect of different [Ca2+ ]i on proliferation and apoptosis in SW480 cells. Methods Immunofluorescence staining was used to detected the localization of CacyBP/SIP treated with inonmycin in SW480 cells. The cell survival rate was detected by methyl thiazol tetrazolium (MTT). Cell cycle and apoptosis was detected by flow cytometry. Results Different concentration of ionomycin were treated with colon cancer SW480 cells and probed by immunocytochemistry staining using anti-CacyBP/SIP antibodies. CacyBP/SIP was distributed throughout the cytoplasm in untreated cells. In ionomycin-treated cells, CacyBP/SIP was also detected in the cytoplasm treated with 1 μmol/L and 2 μmol/L ionomycin. However, 5 μmol/L ionomycin caused a significant translocation of endogenous CacyBP/SIP from the cytoplasm to nucleus. While 10 μmol/L of ionomycin induces CacyBP/SIP retranslocation to cytoplasm and nucleus. The ionomycintreated cells showed no significant effect on cell proliferation by the MTT assay (P=172.918). The results of the cell apoptosis analysis by FACS similarly indicate that ionomycin has no effect on the SW480 cells early apoptosis rate [(0.747±0.049)%] and total apoptosis [(2.383±0.068)%, P=79.618]. Conclusion Although elevating [Ca2+ ]i by ionomycin has affected on the nuclear translocation of CacyBP/SIP, it has no affected on the proliferation and apoptosis in SW480 cells. Key words: Colon cancer; Calcium; Nuclear translocation; Proliferation; Apoptosis

Eomesodermin promotes interferon-γ expression and binds to multiple conserved noncoding sequences across the Ifng locus in mouse thymoma cell lines.

The T-box transcription factors T-bet and eomesodermin (Eomes) have been shown to regulate the lineage-specific expression of interferon-γ (IFN-γ). However, in contrast to T-bet, the role of Eomes in the expression of IFN-γ remains unclear. In this study, we investigated the Eomes-dependent expression of IFN-γ in the mouse thymoma BW5147 and EL4 cells, which do not express T-bet or Eomes. The ectopic expression of Eomes induced BW5147 and EL4 cells to produce IFN-γ in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin (IM). In BW5147 cells, Eomes augmented luciferase activity driven by the Ifng promoter encoding from -2500 to +113 bp; however, it was not increased by a stimulation with PMA and IM. A chromatin immunoprecipitation assay showed that Eomes bound to the Ifng promoter and conserved noncoding sequence (CNS) -22 kb across the Ifng locus with high efficacy in BW5147 cells. Moreover, Eomes increased permissive histone modifications in the Ifng promoter and multiple CNSs. The stimulation with PMA and IM greatly augmented Eomes binding to CNS-54, CNS-34, CNS+19 and CNS+30, which was inhibited by FK506. These results indicated that Eomes bound to the Ifng promoter and multiple CNSs in stimulation-dependent and stimulation-independent manners.

188 HAPLOID ACTIVATION OF BOVINE OOCYTES WITH IONOMYCIN AND SINGLE OR COMBINED ACTIVATING AGENTS

Haploid activation of bovine oocytes is important for reproductive technologies such as intracytoplasmic sperm injection (ICSI) or somatic cell nuclear transfer (SCNT). Nevertheless, it is still a highly inefficient procedure. The aim of this work was to combine different activation drugs, known to have different targets along the activation cascade, to find a more effective activation protocol. Cumulus-oocyte complexes (COC) were aspirated from slaughtered ovaries and in vitro-matured (IVM) for 22 h. Oocytes were activated with 5 µM ionomycin (IO) for 4 min and then randomly allocated into 1 of the following treatments: 50 µM roscovitine (ROSC), 10 µg mL–1 cycloheximide (CHX), ROSC and 10 µM PD0325901 (ROSC/PD), or CHX and PD (CHX/PD) for 5 h; 15 µM dehydroleucodine (DHL) or DHL and ROSC (DHL/ROSC) for 3 h; DHL and CHX for 3 h followed by 2 h with CHX; 5-min exposure to 7% ethanol 4 h post-IO (ET); or ET followed by ROSC (ET-ROSC). Controls were IO followed by 3 h of exposure to 1.9 mM 6-DMAP with or without a previous 3-h culture in TCM-199 (3 h in DMAP and DMAP, respectively). Embryos were cultured in SOF medium. Pronuclear formation (PN) and second polar body extrusion (2PB) were assessed by 5 µg mL–1 propidium iodide oocyte staining, 17 h after IO. Activation was defined as the presence of at least 1 PN, and 2PB extrusion rate was calculated regardless of the nuclear stage. Data were analysed by Fisher’s Test (P &lt; 0.05). Activation (Table 1) was similar in all groups, with the exception of ROSC/PD and ET-ROSC that were the highest and DHL that was the lowest. Although ROSC or CHX seemed to improve 2PB rate when combined with DHL, cleavage decreased significantly, suggesting DHL itself, or its combination with these drugs, negatively affects embryo development. Group ET showed activation rates comparable to other treatments, but it was not reflected on cleavage, suggesting that ET induces PN formation but it might be inefficient to trigger embryo development. Nevertheless, this observation was not made for ET-ROSC, as it showed a higher cleavage rate than ET and ROSC alone. The mitogen-activated protein kinase (MAPK) inhibitor PD showed different effects when combined with ROSC or CHX, despite that they both act on the mammary fat pad (MPF). In ROSC/PD, a slight improvement was observed on activation and cleavage rates compared with ROSC. Group CHX/PD resulted in a slightly higher 2PB percentage, but a lower activation percentage that derived in a significantly lower cleavage than CHX. In conclusion, ROSC and CHX were the most effective single treatments for haploid activation. Moreover, some combined treatments, namely DHL/ROSC and DHL/CHX, proved to be as effective or better at 2PB extrusion rate, which is the defining feature in haploid activation. Table 1.Activation, second polar body extrusion (2PB) and cleavage of bovine oocytes activated with ionomycin followed by single or combined activating agents1

Changes of microRNA-206 in peripheral blood mononuclear cells from children with respiratory syncytial virus bronchiolitis

Objective To explore the role of microRNA-206(miR-206) in peripheral blood mononuclear cells(PBMCs) in infantile bronchiolitis caused by respiratory syncytial virus(RSV). Methods Thirty-five cases of infantile bronchiolitis and 25 cases of healthy controls were enrolled into the current study.PBMCs were isolated from the peripheral blood of both healthy subjects and those with infantile bronchiolitis in the acute and the convalescent stages.Total RNAs were extracted from PBMCs which were stimulated by phorbol-12-myristate-13-acetate(PMA) and Ionomycin, and then the RNA was transcribed reversely into cDNA.The expressions of miR-206 and Kruppel-like transcription factor 4(KLF4) were detected by real-time quantitative polymerase chain reaction(qRT-PCR) method.Plasma interleukin-17(IL-17) was determined by enzyme linked immunosorbent assay(ELISA). Results There was a significant difference in miR-206 levels of children with RSV bronchiolitis in the acute stage(0.055±0.018) and the convalescent stage(0.187±0.069) as well as the healthy controls(0.204±0.075). Through pairwise comparison, the miR-206 levels in the children in the acute stage were significantly lower than those in the convalescent stage and healthy control group(P 0.05). The levels of KLF4 mRNA of children in the acute stage, convalescent stage as well as the healthy subjects were 0.588±0.161, 0.086±0.024, 0.075±0.019, respectively, which was significantly difference(P 0.05). Furthermore, the result of this study showed a negative correlation between the expression of miR-206 and KLF4(r=-0.624, P<0.01)and IL-17(r=-0.609, P<0.01) in children in the acute stage and a positive correlation between KLF4 mRNA and IL-17 in children in the acute stage(r=0.662, P<0.01). Conclusion The levels of miR-206 may play a role in the onset of RSV associated post-bronchiolitis(PB) and the low expression of miR-206 in children infected with RSV may increase the susceptibility to PB. Key words: Capillary bronchitis; Respiratory syncytial virus; MicroRNA-206; Kruppel-like transcription factor 4; Interleukin-17; Infant; Child

Relationship between AKT and PKCθ and morphine-induced inhibition of differentiation of T helper cells

Objective To evaluate the relationship between protein kinase B (AKT) and protein kinase (PKCθ) and morphine-induced inhibition of differentiation of T helper (Th) cells.Methods Peripheral venous blood samples were taken from 20 healthy volunteers..Peripheral blood mononuclear cells (PBMCs) were extracted by density gradient centrifugation method.CD4+ T lymphocytes extracted were purified by magnetic bead separation.CD4+ T lymphocytes were randomly assigned into 5 groups (n =3 each) using a random number table.CD4 + T lymphocytes were incubated routinely in group C.CD4 + T lymphocytes were incubated in the presence of PMA 25 ng/ml + ionomycin 1 μg/ml (group PI),PMA 25 ng/ml + ionomycin 1 μμg/ml + morphine 50 μμg/ml (group M),or PMA 25 ng/ml + ionomycin 1 μμg/ml + morphine 50 μg/ml + naloxone 50 μμg/ml (group N).The cells were incubated for 4 h in the incubator containing 5％ CO2 at 37 ℃.The expression of interferon (IFN)-γ and interleukin-4 (IL-4) was detected by flow cytometry.The expression of IFN-γ and IL-4 was used to reflect the percentage of Th1 and Th2 cells,respectively.The ratio of Th1/Th2 was calculated.The expression of AKT,phosphorylated AKT (p-AKT),PKCθ and phosphorylated PKCθ (p-PKCθ) was detected by Western blot,and the ratio of p-AKT/AKT and p-PKCθ/PKCθ was calculated to reflect the activities of AKT and PKCθ,respectively.Results Compared with group C,the percentage of Th1 and Th2 cells and ratio of Th1/Th2 were significantly increased in PI,M and N groups,the activities of AKT and PKCθ were increased in PI and N groups,and the activity of PKCθ was increased in group M (P ＜ 0.05).Compared with group PI,the percentage of Th1 cells,ratio of Th1/Th2 and activity of AKT were significantly decreased in group M,the ratio of Th1/Th2 was decreased in group N (P ＜0.05),and no significant change in the activity of PKCθ was found in M and N groups (P ＞ 0.05).Compared with group M,the percentage of Th1 cells,ratio of Th1/Th2 and activity of AKT were significantly increased (P ＜ 0.05),while no significant change in the activity of PKCθ was observed in group N (P ＞ 0.05).Conclusion The mechanism by which morphine inhibits the differentiation of Th cells through activating opioid receptors is related to inhibition of AKT activation,but not related to PKCθ. Key words: Morphine; T-lymphocytes, helper-inducer; Protein-serine-threonine kinases; Protein kinase C

Effect of tacrolimus and Staphylococcus aureus enterotoxin B on T helper 17 cells in peripheral blood from patients with atopic dermatitis

Objective To determine the proportion of T helper 17 (Th17) cells in peripheral blood mononuclear cells (PBMCs) from children with acute atopic dermatitis (AD),and to evaluate the effect of tacrolimus and Staphylococcus aureus enterotoxin B (SEB) on the proportion.Methods PBMCs were separated from 86 children with moderate to severe acute AD and 81 healthy children,and cultured in the presence of phorbol ester (PMA) and ionomycin with or without tacrolimus or SEB for different durations.Then,flow cytometry was performed to determine the proportions of Th17,Th1 and Th2 cells,enzyme-linked immunosorbent assay (ELISA) to measure the levels of interleukin (IL)-17,interferon (IFN)-γand IL-4 in the culture supernatant of these cells,and real-time fluorescence-based quantitative PCR (RT-PCR) to detect the expression level of RORγt mRNA.Statistical analysis was done by using two independent samples t-test,chi-square test and analysis of variance.Results After stimulation with PMA and ionomycin,there was a significant increase in the proportions of Th2 and Th17 cells in,supernatant levels of IL-17 and IL-4 and mRNA expression level of RORγt of,but a significant decrease in Th1 cell percentage in and IFN-γ supernatant level of,PBMCs from patients with AD compared with PBMCs from the healthy controls (all P ＜ 0.01).The combination treatment with tacrolimus significantly reduced the supernatant levels of IL-17,IFN-γ,IL-4 and RORγt mRNA expression level of PBMCs from both the patients and healthy controls compared with those treated with PMA and ionomycin only (all P ＜ 0.01).In comparison with the healthy controls,the patients with AD showed significantly higher proportions of Th17 and Th2 cells in,supernatant levels of IL-17 and IL-4 of,and expression level of RORγt mRNA of,but significantly lower proportion of Th1 cells in and IFN-γ supernatant level of,PBMCs after SEB treatment (all P ＜ 0.01).Conclusions Tacrolimus evidently inhibits the secretion of IL-17,IFN-γ and IL-4 by PBMCs from both children with AD and healthy children,while SEB can up-regulate the proportion of peripheral blood Th17 cells in children with AD. Key words: Dermatitis, atopic; Tacrolimus; Staphylococcus aureus; Enterotoxins; Th17 cells

Cytokine signatures of human whole blood for monitoring immunosuppression.

How to evaluate status of the immune system is extremely critical for clinical immunosuppressive treatment. In this study, we tested the secretion of cytokines in undiluted whole blood samples stimulated with Phorbol 12-myristate 13-acetate (PMA) and ionomycin (IONO), and compared the effects of dexamethasone (DEX), cyclosporine A (CsA) or mycophenolic acid (MPA), either alone or in combination, on cytokine profiles. The results showed that both DEX and CsA dose-dependently inhibited the production of eleven cytokines: interleukin (IL)-2, IL-4, IL-5, IL-6, IL-13, IL-17, interferon γ (IFN-γ), tumor necrosis factor α (TNF-α), granulocyte-macrophage colony-stimulating factor (GM-CSF) and granulocyte colony-stimulating factor (G-CSF). Unexpectedly, MPA showed no obvious influences except for the mild inhibition on GM-CSF production. In combination treatment, cytokine profiles reflect not only the synergistic effects among drugs, but also the specific effect of the individual drug. Thus, the effects of different immunosuppressants could be reflected through their specific cytokine signatures, which can be applied to maximize immunosuppressive effects, while to minimize risk of infections and help physicians to reasonably apply immunosuppressants.